Over the last year we have used advanced methods in cryo-electron tomography to image two types of intact bacterial cells that display novel modes of motility. Cells of Flavobacterium johnsoniae, and of many other members of the phylum Bacteroidetes, move rapidly over surfaces in a process known as gliding motility. F. johnsoniae cells typically move at speeds of 2 to 5 m/s over glass surfaces. They also adsorb added latex spheres and propel these around the cell in multiple path. Numerous behavioral, biochemical, electron microscopic and genetic analyses of F. johnsoniae have been conducted to understand gliding but the structures that comprise the motility machinery and the mechanism of cell movement are not known. Using cryo-electron tomography, we showed that wild-type cells display tufts of 4 nm-wide cell surface filaments that appear to be anchored to the inner surface of the outer membrane. These filaments are absent in cells of a nonmotile gldF mutant, but are restored upon expression of plasmid-encoded GldF, a component of a putative ATP-binding-cassette transporter. In related studies on the predatory bacterium Bdellovibrio bacteriovorus, we demonstrated that B. bacteriovorus cells are capable of substantial flexibility and local deformations of their outer and inner membranes without loss of cell integrity. These shape changes can occur in less than 2 minutes, and analysis of the internal architecture of highly bent cells shows that the overall distribution of molecular machines and the nucleoid is similar to those seen in moderately bent cells. B. bacteriovorus cells appear to contain an extensive internal network of short and long filamentous structures. We have suggested that rearrangements of these structures, in combination with the unique properties of the cell envelope may underlie the remarkable ability of B. bacteriovorus cells to find and enter bacterial prey. (see Liu et al (2007) and Borgnia et al (2008) for more details). We have also used electron tomography to analyze structures of nanoparticles such as the icosahedral pyruvate dehydrogenase (PDH) enzyme complex and the cancer drug Doxil. We have shown that electron tomography can be a powerful method for providing quality control on the physical characteristics of complex nanomedicine formulations such as Doxil. We have also shown that individual PDH complexes can be imaged and interpreted in terms of the atomic structures of the E1, E2 and E3 components, further establishing an important role for tomography as a structural tool in nanomedicine. (see Lengyel, Milne et al (2008) and Lengyel, Stott et al (2008) for more details). Over the last year, we have used ion abrasion scanning electron microscopy to show that MNT-1 melanoma cells can be rapidly imaged at resolutions of 30-nm in the z-direction (direction of section removal), and 6 nm in the x-y plane (plane of section removal). We also showed that individual gold and quantum dot particles can be localized in the images, demonstrating that ion-abrasion scanning electron microscopy is a powerful method for obtaining combined information on 3D ultrastructure and molecular localization. In particular, statistical analysis of information obtained from the imaging such as size, shape and compositional analysis of organelles could provide valuable diagnostic markers for discriminating normal cells from abnormal cells. Yet another aspect of these studies concerns applications for clinical and pre-clinical imaging of tissue specimens. Nanoparticles such as gold and iron oxide-based compounds that have electron dense features are especially amenable to detection as imaging agents. We are using ion-abrasion scanning electron microscopy to provide rapid feedback on subcellular localization of these nanoparticles in an effort that could be highly relevant in clinical contexts to determine useful doses, efficiency of tissue targeting, and efficacy of drug delivery to the correct targets. Knowledge of drug distribution could also lead to ideas for chemical modifications that could improve delivery of these nanoscale reagents.